Conversion of Glucosinolates to Isothiocyanates in Humans After Ingestion of Cooked Watercress1

Vol. 8, 447–451, May 1999 Cancer Epidemiology, Biomarkers & Prevention 447

Conversion of Glucosinolates to Isothiocyanates in Humans after Ingestion of Cooked Watercress1

Serkadis M. Getahun and Fung-Lung Chung2 vegetables. Furthermore, upon incubation of the cooked Division of Carcinogenesis and Molecular Epidemiology, American Health watercress juice with fresh human feces under anaerobic Foundation, Valhalla, New York 10595 conditions, ϳ18% of glucosinolates was hydrolyzed to ITCs in 2 h. These results suggest that the microflora in the intestinal tract are a likely source for the hydrolysis Abstract of glucosinolates to ITCs in humans. Isothiocyanates (ITCs), major constituents of cruciferous vegetables, can inhibit tumorigenesis in rodents by Introduction modulating the metabolism of carcinogens. ITCs that ITCs,3 which occur as major constituents in cruciferous vege- occur as glucosinolates are released by myrosinase- tables, have been shown to inhibit tumorigenesis in rodents mediated hydrolysis when raw vegetables are chopped or treated with carcinogens (1–4). Studies in vitro and in vivo chewed. However, because cruciferous vegetables are showed that the inhibitory activity of ITCs against carcinogen- commonly consumed by humans after being cooked, it is esis can be attributed to their ability to deactivate phase I important to examine whether dietary glucosinolates are enzymes and/or to activate phase II enzymes (3, 5). Recent converted to ITCs after cooked cruciferous vegetables in studies in humans also demonstrated that the consumption of which myrosinase is deactivated have been consumed. cruciferous vegetables resulted in an increase in glutathione This information is useful for evaluating the potential transferase activity and a decrease in the metabolic activation of role of ITCs in cruciferous vegetables in the protection carcinogens in smokers (6–8). These results support a potential against human cancers. A urinary marker, based on a role of ITCs in cruciferous vegetables in the prevention of cyclocondensation product formed by the reaction of human cancers. ITCs and their conjugates with 1,2-benzenedithiol, was ITCs are products of degradation of glucosinolate precur- used to quantify the uptake of dietary ITCs in humans. sors in cruciferous vegetables, a reaction usually catalyzed by At breakfast and lunch, nine volunteers consumed a total myrosinase, an enzyme that is activated upon crushing the of 350 g of cooked watercress in which the myrosinase vegetables by chopping or chewing (Fig. 1a; Refs. 9 and 10). activity was completely deactivated. On the basis of the We and others have demonstrated that significant amounts of analysis of ITCs in the cooked watercress upon adding ITCs are released after ingesting raw cruciferous vegetables, as exogenous myrosinase, the amount of glucosinolates ␮ indicated by the presence of ITC mercapturic acid in the urine ingested by each subject was estimated to be 475 mol. (11–13). Little is known, however, about the fate of glucosi- The 24-h urine samples showed that the total urinary nolates in humans upon consumption of cooked vegetables in excretion of ITC conjugates in the subjects ranged from ␮ which the myrosinase activity is absent. Because crucifers are 5.6 to 34.8 mol, corresponding to 1.2–7.3% of the total commonly consumed by humans after being cooked, it is im- amount ingested. On the basis of our previous results ϳ portant to know whether glucosinolates in cooked vegetables that 50% of dietary ITCs were excreted in the urine as are converted to ITCs. This information can be useful in the conjugates, these values represent the minimal in vivo investigation of the potential roles of dietary ITCs in human conversion of glucosinolates to ITCs. For purposes of cancers. Here, we studied the metabolic conversion of glucosi- comparison, we carried out a second experiment in which nolates to ITCs in humans after ingesting cooked and uncooked 150 g of uncooked watercress were consumed. The watercress and compared the extent of conversion. Because percentage of urinary ITC conjugates excreted in this ITCs are primarily metabolized via the mercapturic acid path- study ranged from 17.2 to 77.7% of the total ingested way, the N-acetylcysteine conjugate of ITC in the urine serves ITCs. These results indicate that glucosinolates are as a marker of ITCs released from glucosinolates. Using a converted to ITCs in humans after ingestion of cooked previously developed assay based on the reaction of ITCs or watercress, in which the myrosinase has been completely their thiol conjugates with 1,2-benzenedithiol forming a cyclic inactivated. The extent of conversion, however, is dithiol thione product (Fig. 1b; Refs. 14 and 15), we quantified considerably less than that after ingesting uncooked the urinary metabolites of ITCs after eating watercress with and without myrosinase activity. In addition, we also examined whether intestinal microflora are a possible source for the Received 10/7/98; revised 2/22/99; accepted 3/8/99. degradation of glucosinolates in humans by incubating cooked The costs of publication of this article were defrayed in part by the payment of watercress juice with human fecal samples. page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by NCI Grant CA46535. This is Paper No. 30 in the series “Dietary Inhibitors of Chemical Carcinogenesis.” 2 To whom requests for reprints should be addressed, at American Health Foun- dation, 1 Dana Road, Valhalla, NY 10595. Phone: (914) 789-7161; Fax: 3 The abbreviations used are: ITC, isothiocyanate; HPLC, high-performance (914) 592-6317; E-mail: [email protected]. liquid chromatography.

Table 1 Cumulative amounts of ITC conjugates in 24-h human urine before (baseline) and after ingestion of cooked watercress meal

Table 2 Experimental Protocol

Days 1 and 2 Day 3 Day 4 Baseline Watercress consumption Follow-up

urine sample was collected on day 2 as baseline prior to the consumption of watercress on day 3. Fresh watercress (Nas- turtium officinale) purchased from a local supermarket was cooked in boiling water for 3 min (85.7 g/liter), and a total of 350 g was ingested by each volunteer, 200 g at breakfast and 150 g at lunch. After breakfast, a 27-h urine sample was collected from each subject. Another 18-h urine sample was collected after lunch on day 4 as the follow-up. After a 7-day period, a second experiment was conducted. The same protocol was used with the exception that uncooked watercress was consumed. Because we have previously conducted a similar study using uncooked watercress (12), in this experiment, only four subjects (subjects 1, 2, 7, and 8; see Table 3) participated who consumed 100 g for breakfast and 50 g for lunch on day 3. In both experiments, a 10-ml aliquot of each pooled urine sample was stored at Ϫ20°C until ITC analysis. Analysis of ITC in Urine. The amount of ITCs in the urine Fig. 1. a, myrosinase-catalyzed conversion of glucosinolates to ITCs via Lossen samples was analyzed by a previously described method (15). rearrangement. b, reaction of ITC with 1,2-benzenedithiol forming the cyclocondensation product, 1,3-benzodithiole-2-thione, and amine. Briefly, the urine sample was thawed and vortexed. A 1-ml aliquot was placed in a 2-ml centrifuge tube and centrifuged for 20 min at 1000 ϫ g. An aliquot (100 ␮l) of the supernatant was mixed with 600 ␮l of 1,2-benzenedithiol in 2-propanol (10 mM, Materials and Methods degassed) and 500 ␮l of phosphate buffer (0.1 M, pH 8.5, Chemicals. 1,2-Benzenedithiol was purchased from Lancaster degassed) in a 2-ml Chromacol autosampler vial with a screw Synthesis, Inc. (Windham, NH). 1,3-Benzodithiole-2-thione cap (Chromacol, Inc., Trumbull, CT). The mixture was then was prepared and characterized according to a published vortexed for 1 min and incubated at 65°C for2hinawater bath method (14, 15). Myrosinase was prepared from mustard seeds shaker. The product 1,3-benezodithiol-2-thione was analyzed ␮ ϫ according to a published procedure (16). Nutrient broth me- by reverse-phase HPLC using a Waters Bondapak C18 (150 ␮ dium was purchased from Difco Laboratories (Detroit, MI). 3.9 mm) with a C18 Waters Bondapak guard column and a Sodium thioglycolate was purchased from Fisher Scientific detection wavelength of 365 nm. The mobile phase consisted of

(Fair Lawn, NJ). All other chemicals and solvents were reagent a mixture of methanol and H2O (7:3 v/v) with a flow rate of grade and were obtained from commercial sources. 1.75 ml/min. A standard curve was constructed using a series of Study Protocol. Nine healthy volunteers (designated 1–9; five 1,3-benzodithiole-2-thione solutions with concentrations rang- females and four males; Table 1) ages 23–45 years were ing from 0 to 20 ␮M in 2-propanol:water (1:1 mixture). Each recruited for the study. All subjects signed a consent form urine sample was aliquoted three times and analyzed in tripli- before participating in the 4-day study. The experimental pro- cate. A negative control sample contained only deionized water. tocol is outlined in Table 2. Analysis of ITC in Watercress. The cooked watercress (113 Volunteers were asked to avoid foods containing ITCs g) was blended for 2 min in deionized water (100 ml) using a such as mustard and cruciferous vegetables, including broccoli, Mini-Prep blender (Cuisinart) until it became a fine paste. The cabbage, horseradish, turnips, brussels sprouts, collard greens, paste was then poured through six layers of cheesecloth, and the and tender greens, for the entire experimental period. A 24-h liquid was squeezed into a 500-ml beaker. Another portion of

Table 3 Cumulative amounts of ITC conjugates in 24-h human urine before (baseline) and after ingestion of uncooked watercress meala

Urine excreted before ITCs in baseline urine Urine excreted after ITCs in urine after % of administered dose excreted Subject no. Sex consumption (liters) (␮mol)a consumption (liters) consumption (␮mol)a as ITC conjugatesb 2 F 1.4 0.7 Ϯ 0.0 2.0 755.2 Ϯ 0.4c 77.7 1 M 2.0 1.6 Ϯ 0.0 3.2 447.3 Ϯ 0.5 46.3 7 F 1.4 0.7 Ϯ 0.1 2.8 388.0 Ϯ 2.4 39.6 8 M 2.5 1.7 Ϯ 0.1 1.9 167.2 Ϯ 0.1 17.2 a Mean Ϯ SD (triplicate data). b On the basis of 972 ␮mol of ITCs in uncooked watercress extract. c Values obtained after subtracting the baseline concentration. deionized water (125 ml) was added, and the liquid was again phosphate buffer pH 8.5, the mixture was incubated at 65°C squeezed into the beaker. This process was repeated a third for 2 h. The cyclocondensation reaction product was ana- time, and the combined liquid (350 ml) was transferred to a lyzed by HPLC, as described above. 500-ml volumetric flask and deionized water was added to 500 ml. A 10-ml aliquot of this solution was then filtered through Whatman filter paper no. 1 using a Buchner funnel to obtain a Results and Discussion clear solution. One ml of myrosinase (2 mg/ml) in 0.1 M We have previously shown that the cyclocondensation product potassium phosphate buffer (pH 6.6) was then added to the 1,3-benzodithiol-2-thione provides a useful and specific urinary filtrate; the mixture was then incubated at 37°C for 2 h. For the biomarker for quantifying uptake of ITCs after ingesting un- analysis of ITCs released in the incubation mixture, 100 ␮lof cooked watercress and mustard (15). Because the ITC conju- the myrosinase-treated watercress juice were subjected to the gates are in equilibrium with free ITCs and can react with thiols cyclocondensation reaction as described above. The glucosino- through an exchange reaction, the mercapturic acid conjugates lates and free ITCs in the broth after cooking watercress were excreted in the urine readily undergo the cyclocondensation also determined by the same method. reaction with 1,2-benzenedithiol (15, 17). Using this assay, we For the determination of ITC content of uncooked wa- demonstrated that thiol conjugates of ITCs were excreted in the tercress, 10 g was finely chopped in Mini-Prep blender, urine after consumption of cooked watercress. These results followed by the addition of methanol (50 ml). In this exper- provide clear evidence that glucosinolates in cooked vegetables iment, methanol was used for efficient extraction of ITCs. are converted to free ITCs in humans. The mixture was then blended into a fine paste. The resulting The cooked watercress used in this study was completely paste was filtered through six layers of cheesecloth, and the devoid of myrosinase activity, as indicated by the absence of residue was again washed with 50 ml of methanol. The ITCs in the juice of the homogenized watercress. Upon addition procedure was repeated two more times, and the final vol- of exogenous myrosinase to the cooked watercress juice fol- ume was adjusted to 200 ml in a volumetric flask. A 10-ml lowed by incubation at 37°C for 2 h, 1.4 ␮mol of ITCs per 1 g aliquot of this solution was filtered through a Whatman filter of vegetable were released. It is estimated, based on this value, paper no. 1 using a Buchner funnel; a 100-␮l aliquot of the that a total of 475 ␮mol of ITCs were consumed by each subject filtrate was subjected to the cyclocondensation reaction as after eating 350 g of cooked watercress. The amount of my- described above. rosinase used and the incubation time were determined by Release of ITCs from Cooked Watercress Juice upon Incu- monitoring the formation of ITCs from known concentrations bation with Human Feces. A fresh human fecal sample was of sinigrin ranging from 0.7 ␮M to 0.4 mM. Under these con- stored in a disposable anaerobic system (Gas pak; Becton ditions, the conversion of sinigrin to allyl ITC was quantitative Dickinson Co., Cockeysville, MD) with a hydrogen-carbon at all concentrations examined. Therefore, the amount of 475 dioxide generator container. The stool specimen (20 g) was ␮mol is likely to represent the total glucosinolate content in the placed in a 50-ml centrifuge tube containing 15 ml of 0.01% cooked watercress because increasing the incubation time or sodium thioglycolate in 50 mM sodium phosphate (pH 7.2). amount of myrosinase did not result in further hydrolysis. The above mixture was vortexed under nitrogen in a flow Gluconasturtiin, the precursor of phenethyl ITC, constitutes hood and centrifuged at 2000 ϫ g for 5 min. The supernatant Ͼ30% of the total glucosinolate content in watercress (12). The was placed in another tube, and the sediment was vortexed glucobrassicins, minor components in watercress that are con- with an additional 30 ml of buffer. The supernatants were verted to indoles, do not hydrolyze to ITC by myrosinase (10). combined and sonicated at 0°C for 30 s and centrifuged In this study, we noted a 6-fold difference in the total levels of again at 2000 ϫ g for 30 min. A 3-ml aliquot of the ITC metabolites excreted in the 24-h urine collected from each supernatant was mixed with 15 ml of sterilized nutrient broth of the nine subjects, ranging from 5.6 to 34.8 ␮mol after media (0.65%, pH 7.2) and 2 ml of cooked watercress juice. subtracting the background levels (Fig. 2 and Table 1). The The mixture was placed in a serum bottle and sealed with a background levels of cyclocondensation product found in base- rubber stopper and an aluminum cap. The serum bottle was line urine samples are intriguing. Similar background levels incubated at 37°C with agitation for 30 min and 1, 2, 3, 4, were found in the urine samples collected during the follow-up and 6 h. As negative control, the fecal homogenate was period (day 4). It is not clear whether they come from residual boiled at 100°C for 10 min. In addition, 2 ml of watercress ITCs or other related compounds. On the basis of the total juice were incubated under identical conditions with 1 ml of glucosinolates (475 ␮mol) consumed, the rate of conversion to myrosinase (2 mg/ml) and 17 ml of nutrient broth for2has ITC ranged from 1.2 to 7.3%. If one assumes that ϳ50% the positive control. The incubation was terminated by mix- administered ITCs are excreted in the 24-h urine as reported ing 500 ␮l of the incubation mixture with 800 ␮lof10mM previously (12, 13), this represents a total conversion of 2.4– 1,2-benzenedithiol in 2-propanol. After addition of 500 ␮lof 14.6%.

Fig. 3. The percentage of glucosinolates converted to ITCs upon incubation with human fecal homogenates.

loss of glucosinolates in cruciferous vegetables (18). In the experiment with the uncooked watercress, the amount of ITC metabolites in the 24 h urine was found to range from 167 to 755 ␮mol, equivalent to a conversion rate from 17.2 to 77.7% (Table 3). These rates of conversion are comparable to those reported previously for phenethyl and allyl ITCs, which ranged from 30 to 67% (12, 13). A 4-fold difference in the metabolism of dietary ITCs among the subjects in this experiment was observed. These results indicate that the extent of conversion in humans after consumption of the uncooked watercress was at least 10-fold greater than that of the cooked watercress. Inter- estingly, in both experiments, subjects 1 and 2 had a higher level of urinary metabolites than those found in subjects 7 and 8, suggesting there may be individual variabilities in the metabolism of ITCs via the mercapturic acid pathways. Oginsky et al. (19) previously showed that goitrin was detected in urine and blood of humans after oral administration of pure progoitrin, the glucosinolate precursor of goitrin. This group reported that a variety of bacterial species, including those present in human feces, appeared to have myrosinase-like activity (20). Goodman et al. (21) described thioglucosidase activity in the tissues of mammals, including humans. Our study is the first to show that the intact glucosinolates in cooked cruciferous vegetables can be converted to ITCs in humans. To Fig. 2. Reverse-phase HPLC chromatograms obtained from analysis of a 24-h verify the potential role of intestinal microflora in the conver- human urine sample after consumption of cooked watercress (a) and raw water- sion, we examined whether human feces possess such activity cress (b). Peak at 3.2 min is identified as 1,3-benzodithiole-2-thione by its under anaerobic conditions. Upon incubating cooked water- retention time and UV spectrum as compared to the synthetic standard. cress juice with human fecal samples, we observed a linear increase of ITC up to 17.7% for the first2hofincubation. No further increase of ITCs was seen after 2 h, possibly due to the To compare the extent of conversion in humans after ITC-mediated inhibition of enzyme activities (Fig. 3). ITCs ingesting cooked and uncooked watercress, we carried out a were not detected in the incubations with the heat-deactivated second experiment in which four subjects who participated in fecal homogenate. These results demonstrated that a myrosi- the first experiment with cooked watercress were asked to eat nase-like activity is present in human feces, supporting the a total of 150 g of uncooked watercress for breakfast and lunch. notion that microflora in the gut may contribute to the degra- The total ITCs in the 150 g of uncooked watercress were 972 dation of glucosinolates to ITCs in humans. We cannot, how- ␮mol or 6.5 ␮mol/g, as compared to 1.4 ␮mol/g in the cooked ever, exclude the possibility that nonspecific thioglycosidase watercress after myrosinase treatment. A substantial amount of activity is present in tissues (21). glucosinolates was found in the broth (2.8 ␮mol/g) of the Studies have shown that glucosinolates inhibited tumori- cooked watercress after myrosinase treatment. These results are genesis in carcinogen-treated rodents (22, 23). The mecha- consistent with the notion that cooking may cause a significant nism(s) by which glucosinolates inhibited tumorigenesis is not

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Serkadis M. Getahun and Fung-Lung Chung

Cancer Epidemiol Biomarkers Prev 1999;8:447-451.

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